基于常压室温等离子体诱变和高通量筛选选育L-色氨酸高产菌株

Breeding of L-tryptophan high-yield strain by ARTP mutagenesis and high-throughput screening

摘要:

L-色氨酸是人体中不可或缺的必需氨基酸，由于其广泛的应用和国内外巨大的需求，L-色氨酸已成为备受关注的研究和产业发展方向。尽管非理性诱变育种策略是一种开发工业菌株的有效手段，但是如何筛选具有理想表型的菌株依然是一个重大挑战。为了提高筛选L-色氨酸高产菌株的效率与准确性，本研究通过常压室温等离子体诱变构建随机突变文库，并结合深孔板高通量筛选，基于能够特异性响应L-色氨酸的拟荧光蛋白传感器从随机突变文库中成功筛选出一株L-色氨酸高产菌株，其产量在摇瓶中达到1.99 g/L，较出发菌株提高了41.77%。还通过基因组与转录组的比较组学分析，进一步对菌株的高产机理进行了解析。本研究基于常压室温等离子体诱变和高通量筛选策略，成功筛选出L-色氨酸高产菌株，为后续进一步选育和开发相关优质的L-色氨酸生产菌株资源提供了坚实的研究基础。

Abstract:

L-tryptophan is an indispensable essential amino acid with a wide range of applications, which leads to a high demand. Accordingly, the production of L-tryptophan becomes a much-anticipated direction in research and industrial development. While irrational mutagenesis is an effective means to breed industrial strains, how to screen the strains with desirable phenotypes is still a major challenge. In order to improve the efficiency and accuracy of screening L-tryptophan high-yield strains, we used atmospheric and room temperature plasma mutagenesis to construct a random mutant library and then combined it with high-throughput screening in deep-well plates. Using a pseudo-fluorescent protein sensor capable of responding specifically to L-tryptophan, we successfully screened out a strain producing L-tryptophan at a high yield from a random mutagenesis library. The fermentation with the strain in shake flasks produced L-tryptophan at a yield of 1.99 g/L, which was 41.77% higher than that of the starting strain. Finally, the mechanism of high yield of the strain was deciphered by comparative genomics and transcriptomics. The above strategies provide a solid research foundation for further selection and development of high quality L-tryptophan producing strains.